bioRxiv preprint doi: ... · 10/5/2020 · Recent evidence suggests MAITs are capable of providing...

Title: A novel subset of follicular helper-like MAIT cells has capacity for B cell help and antibody production in the mucosa.

Author List: Owen Jensen1,2, Shubhanshi Trivedi1, Jeremy D. Meier3, 4, Keke Fairfax2, J. Scott Hale2, Daniel T. Leung1, 2* 1 Division of Infectious Diseases, Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, USA 2 Division of Microbiology & Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, USA 3 Division of Otolaryngology-Head and Neck Surgery, University of Utah School of Medicine, Salt Lake City, USA 4 Primary Children’s Hospital, Salt Lake City, USA * Corresponding author: [email protected] One Sentence Summary: We identified and characterized a novel subset of T follicular helper-like MAIT (MAITfh) cells that has the capacity to provide B cell help, and show the sufficiency of MAIT cells to promote production of pathogen-specific IgA antibodies and B cell differentiation in mucosal challenge.

Abstract

Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes that aid in protection against

bacterial pathogens at mucosal surfaces via release of inflammatory cytokines and cytotoxic molecules.

Recent evidence suggests MAITs are capable of providing B cell help. In this study, we describe a

previously unreported population of CXCR5+ T follicular helper (Tfh)-like MAIT cells, MAITfh, that

have the capacity to provide B cell help within mucosal lymphoid organs. MAITfh cells are preferentially

located near germinal centers in human tonsils and express the classical Tfh-associated transcription

factor, B-cell lymphoma 6 (BCL-6), co-stimulatory markers, inducible T cell costimulatory (ICOS) and

programmed death receptor 1 (PD-1), and cytokines, interleukin (IL)-21. Furthermore, we demonstrate

the ability of MAITs to provide B cell help in vivo following mucosal challenge with Vibrio cholerae.

Specifically, we show that adoptive transfer of MAITs into ab T cell-deficient mice promoted B cell

differentiation and increased serum V. cholerae-specific IgA and bactericidal responses. Our data

demonstrate the capacity of MAITs to participate in adaptive immune responses, and suggest that MAITs

may be potential targets for mucosal vaccines.

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Introduction

Mucosal surfaces are in constant contact with both commensal and pathogenic microbes. One of the

primary means of protection against invading pathogens is the production of secretory IgA and IgM by

plasmablasts (PB) and plasma cells (PC) (1). In the lamina propria of the gut and lung, T independent low

affinity IgA is produced against non-protein antigens by short-lived PBs. Conversely, T dependent high

affinity antibodies against protein antigens are typically generated during germinal center reactions

between T follicular helper (Tfh) and follicular B cells within local lymph nodes (LNs) or mucosal

associated lymphoid tissues (MALT) (2). T follicular helper (Tfh) cells migrate to B cell follicles via

upregulation of CXCR5 and downregulation of CCR7 (3). Within GCs, Tfh cells highly express the

lineage-defining transcription factor, BCL6, and co-stimulatory molecules, PD1, ICOS, and CD40L (4).

PD1 is important for Tfh GC positioning and function (5), while ICOS (6) and CD40L (7) engagement

with B cells are essential for Tfh activation and GC formation (4). Tfh also produce cytokines, such as IL-

21, that promote GC B cell responses (8).

Although Tfh cells are the primary drivers of T dependent GC responses, innate T cells, including

invariant Natural Killer T (iNKT) cells and gd T cells, also have Tfh like subsets capable of B cell help. In

particular, iNKT cells have been well established in mice to provide both cognate and non-cognate help to

B cells (9–18). Murine iNKTfh cells engage in cognate help leading to germinal center formation, and

antibody class switching and production (12, 14, 18). iNKTfh cells have also been shown to promote non-

cognate B cell help by licensing dendritic cells to recruit and activate Tfh cells (10, 11). CXCR5+ gd T

cells can promote antibody production in vitro (19, 20), and promote Tfh differentiation in vivo (21).

More recent evidence suggests that another type of innate-like lymphocyte, Mucosal-associated invariant

T (MAIT) cells, are capable of B cell help (22–26).



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MAIT cells are innate-like ab T cells defined by the expression of an invariant a chain, generally Va7.2

linked to Ja33, 12 or 20 in humans and Va19 linked to Ja33 in mice, and a limited array of TCRb chains

(27–29). MAITs are highly enriched in human blood, liver and mucosa and are appreciated for their rapid

ability to respond to microbial Vitamin B metabolites presented on the MHC class I related protein, MR1,

or cytokine stimulation (30–33). Upon stimulation, MAITs produce pro-inflammatory cytokines

including IFNg, TNFa, and IL-17A and cytotoxic molecules including Granzyme B and Perforin (34–36).

Furthermore, using MAIT deficient mice (MR1-/-), several studies have exemplified the importance of

MAITs in innate immunity against mucosal bacterial pathogens (37–40).

Recent evidence suggests MAITs play a role in adaptive immune responses through B cell help. Analysis

of human peripheral blood MAITs and serum antibody responses following Vibrio cholerae infection (22)

and Shigella vaccination (35), revealed strong associations between MAIT frequency and activation with

polysaccharide-specific IgA and IgG responses, but not with protein antibody responses (22). We have

recently shown that human blood MAITs have the capacity to induce antibody production and B cell

differentiation in vitro, and can secrete the B cell help cytokines following stimulation (23). Analysis of

pleural effusions from tuberculosis patients revealed a population of PD1High MAITs secreting key B cell

help cytokines (24). Furthermore, two recent animal studies demonstrate the importance of MAITs in B

cell help in murine autoimmunity (25) and mucosal vaccine immunity in non-human primates (26).

Our aims for this study were two-fold. We first wanted to determine whether a specific subset of MAIT

cells are responsible for the B cell help phenotype. Our second aim was to determine in vivo if MAITs

were sufficient to induce antibody production and humoral immune protection in the context of mucosal

challenge. We found that like other innate-like T cells, MAITs have a Tfh like subset enriched within

mucosal lymphoid organs. This MAITfh population expresses classical Tfh co-stimulatory markers,

transcription factors, and cytokines, and is localized near B cell follicles. We further show, in a murine



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model, that adoptively transferred MAITs are capable of generating protective antibody responses against

mucosal bacterial pathogens in the absence of other ab T cells. Additionally, we find that in the context

of mucosal challenge, MAITs promote increased production of microbe-specific IgA antibodies and

mucosal B cell differentiation. These results suggest that MAITs have the capacity to enhance mucosal

antibody mediated immunity, and thus may be a prospective target in future mucosal vaccine

development.

Results

CXCR5+ MAITs are increased in tonsils and express higher levels of Tfh co-stimulatory markers

compared to peripheral blood.

With recent evidence highlighting the ability of MAITs to provide B cell help, and studies showing a

modest population of MAITs within human and mouse mucosal lymphoid tissues (39, 41, 42), we aimed

to determine if MAITs had a Tfh-like subset capable of B cell help. To investigate this, we used flow

cytometry to analyze MAIT expression of classical Tfh markers in human peripheral blood mononuclear

cells (PBMCs) and tonsils. We obtained PBMC’s from adult blood donors, and tonsils from children,

ages 2-16, undergoing tonsillectomy for recurrent tonsillitis or tonsillar hyperplasia. MAITs were defined

as CD3+ Va7.2+ MR1-Tetramer+ cells (Fig. 1A). Median MAIT frequency among total CD3+ cells in

tonsils was 0.23% (interquartile range (IQR) = 0.71%, 0.29%) compared to 1.03% (IQR = 0.62%, 1.35%)

among PBMCs (Fig. 1B). We found that a higher percentage (Median=30%, IQR=10.36%, 42.5%) of

tonsil MAITs were CXCR5+ compared to PBMC MAITs (Median=1.74%, IQR=0.37%, 2.16%,

p<0.0001), although there was significant variability among tonsil MAITs (Fig. 1C). We next wanted to

determine if there were differences in Tfh co-stimulatory marker expression between CXCR5+ and

CXCR5- MAIT populations. CXCR5+ MAITs in both tonsils and PMBC’s had significantly higher ICOS

(Fig. 1D) and PD1 (Fig. 1E) expression compared to CXCR5- MAITs of the same tissue. Additionally,

CXCR5+ MAITs in tonsils had significantly higher PD1 and ICOS expression compared to PBMC



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CXCR5+ MAITs. Notably, ICOS and PD1 frequency and fluorescence intensity among tonsil CXCR5+

MAITs are similar to that seen in tonsil CD4+ CXCR5+ (Tfh) cells (data not shown). Taken together, a

high proportion of MAIT cells in tonsils have a CXCR5+ phenotype with Tfh-like co-stimulatory

markers.

Fig. 1. Increased expression of Tfh co-stimulatory molecules in tonsil MAIT cells. (A) Representative FACS plots of unstimulated CD3+ PBMC (top) and tonsil (bottom) cells with MAIT (gated in left panel) co-expression of CXCR5 with ICOS (middle) and PD1 (right). (B) Quantification of MAIT frequency as percentage of CD3+. (C) Frequency of CXCR5+ MAITs. Frequency of (D) ICOS+ and (E) PD1+ MAITs broken down by CXCR5 expression. Data are represented as median from 2 independent experiments. n ³ 13. *p < 0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-tailed Mann-Whitney U test.



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Transcriptional analyses of CXCR5+ MAITs reveals expression of Tfh associated cytokines and

transcription factors

We have previously demonstrated that peripheral blood MAITs can secrete B cell help cytokines IL-6, IL-

10 and low levels of IL-21 in vitro (23). MAITs are also potent producers of IFNg (43), which is known

to have both inhibitory and activating roles in B cell development, proliferation, and antibody responses

(44). In order to investigate the transcription factor and cytokine expression of CXCR5+ and CXCR5-

MAITs, we examined transcript levels of fluorescent associated cell sorted (FACS) MAIT and non-MAIT

CD3+ T cell populations from tonsil and blood. We refer to the sorted populations, as CD8+ (CD8+

CXCR5-), Tfh (CD4+ CXCR5+), MAIT (MR1-Tet+, Va7.2+, CXCR5-), and MAITfh (MR1-Tet+, Va7.2+,

CXCR5+) (fig. S1A). We first assayed for the Tfh lineage-defining transcription factor (TF), BCL6. The

MAITfh population within PBMC and tonsils had higher BCL6 expression compared to MAITs and Tfh

cells. (Fig. 2A). In comparison, analysis of canonical T cell associated TFs TBX21 (Th1) (p=0.03), and

RORC (Th17) (p=0.03), revealed higher expression in tonsil MAITs over the tonsil MAITfh population

(fig. S1B). Both MAIT and MAITfh groups had higher expression of RORC and TBX21 compared to Tfh

groups in PBMCs (MAIT:Tfh p=0.03, MAITfh:Tfh p=0.03) and tonsils (MAIT:Tfh p=0.03, MAITfh:Tfh

p=0.03) (fig. S1B). Given that conventional Tfh cells are known to co-express TFs associated with other

helper T cell subsets based on lineage and environment (45–47), our data suggest that co-expression of

non-follicular helper-associated TFs in the MAITfh population may suggest potential for plasticity in

phenotype or differentiation from a Th1, Th17 or CD8-like subset.



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Fig. 2. CXCR5+ MAITs highly express B cell help cytokine IL-21. (A) Bcl6, (B) Tnfsf13b, (C) Il-6, (D) Il-10, (E) Il-21 qPCR data of unstimulated FAC sorted PBMC and tonsil T cell populations. Data are represented as DDCt relative to an internal housekeeping gene, b-Actin, and the PBMC-CD8+ CXCR5-

population. (F) Representative flow cytometry from PBMC and tonsil T cell populations of IL-21 and IFNg co-expression following 6 h stimulation with PMA/Ionomycin and Brefeldin A for final 4h. (G) Frequency of T cell populations expressing IL-21, IFNg or both. Data are represented median from 2 independent experiments. n³5. (H) IL-21 ELISA following 42 h PMA/Ionomycin stimulation of »40,000 FAC sorted mono-cultured T cell populations described below. Data are Mean with SD, n³3. T cell populations described gated on live CD3+ cells are as follows: Black =CD8+ CXCR5-, Blue = Tfh (CD4+ CXCR5+), Red= MAIT (MR1-Tetramer+ Va7.2+) CXCR5-, Green= MAIT CXCR5+. Representative gating for populations found in Sup. Fig 1a. *p < 0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-tailed Mann-Whitney U test.



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We next assayed for cytokines known to play a role in B cell differentiation or antibody production.

TNFSF13B (BAFF) expression was significantly higher in both MAIT and MAITfh groups within tonsils

and PBMCs compared to Tfh cells in their respective compartments (Fig. 2B). Similar results were seen

in both IL-10 (Fig. 2C) and IL-6 (Fig. 2D) expression with trends of increased expression in MAITfh cells

in PBMCs, though not in tonsils as there was significant variability between donors. Lastly, IL-21

expression in tonsil MAITs and MAITfh, although 2-4 fold lower than tonsil Tfh cells, were highly

elevated compared to corresponding populations in peripheral blood. Furthermore, the PBMC MAITfh

population had a 46-fold and 2-fold enrichment in IL-21 expression compared to the PBMC MAIT and

Tfh populations, respectively (Fig. 2E). Taken together, CXCR5+ MAIT (MAITfh) cells express Tfh-

associated cytokines and transcription factors at levels similar or higher than Tfh cells in their respective

compartments

CXCR5+ MAITs highly express IL-21 compared to CXCR5- MAITs

We next wanted to investigate the relationship between IL-21 and IFNg expression among MAIT subsets

as both cytokines are highly expressed and generally associated with a Tfh vs Th1 like phenotype. When

we stimulated PBMCs with PMA/Ionomycin, we saw a clear dichotomy between IFNg and IL-21 positive

cells in all populations with very low frequency of double positive cells, and the vast majority being

IFNg+ (Fig. 2 F and G). MAITfh had a significantly lower frequency of IFNg+ cells compared to

conventional MAITs (p<0.01). This effect was even more notable in tonsils (p<0.001), where IFNg

production was significantly reduced compared to that of PBMCs (p<0.001), with a similar dichotomy

between IFNg-expressing MAITs and IL-21-expressing MAITfh. Furthermore, the tonsil MAITfh

population had a significantly (p<0.01) higher frequency of IL-21+ cells compared to conventional Tfh

cells following stimulation (Fig. 2F and G). Conversely, IFNg+ frequency is low in all unstimulated

populations but increases drastically in the tonsil MAIT population from >1% to approximately 88%

following stimulation (fig. S2 A and B). To confirm the IL-21 production capacity of MAITs and



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MAITfh cells within tonsils, we sorted MAITs (CD3+ Va7.2+ MR1-Tetramer+ CXCR5-), MAITfh (CD3+

Va7.2+ MR1-Tetramer+ CXCR5+), CD8+ CXCR5- and GC Tfh (CD4+ CXCR5+ PD1high) populations, and

stimulated mono-cultures of these cells with PMA/Ionomycin (48). We found higher IL-21 production in

the MAITfh over MAIT groups and no statistical difference in IL-21 production between MAITfh and

GC Tfh populations (Fig. 2H). In addition, we found an increase in frequency of IL-6 and IL-10 positive

cells following stimulation (fig. S2 C and D), concordant with what was found with qPCR analysis above.

Taken together, these data demonstrate the potent ability of the tonsil MAITfh population to produce B-

cell help cytokines and the clear dichotomy in phenotype between classical MAITs and MAITfh cells in

both peripheral blood and tonsil tissue.

PD1+ MAITs localize near germinal centers within tonsils

Having established that CXCR5+ MAITs exhibit a Tfh-like phenotype in vitro, we next sought to

determine the in vivo spatial differences between CXCR5- and CXCR5+ MAITs in relation to germinal

centers (GCs) in tonsil cryosections. MAITs were defined by co-staining of TRAV1-2 and CD161 as

described in Leng et al. 2019 (49). IgD was used to stain naïve B cells making up the follicular mantle,

and potential IgD+ memory B cells in the marginal or superficial zone (50). Due to difficulties with co-

staining MAITs and CXCR5, we used PD1 to mark MAITfh and non-MAIT GC Tfh cells. PD1 has been

used to mark GC light zone Tfh cells in non-human primates (51), and further analyses of flow cytometry

data (Fig. 1) verify a strong correlation (r=0.9373, p<0.001) between PD1+ and CXCR5+ percent

frequencies in tonsil MAITs (Fig. 3A). Additional analyses confirm that a median of 92% (IQR=78.03%,

92.18%) of PD1+ MAITs are CXCR5+, compared to a median of 15% (IQR=13.18%, 23.1%) for PD1-

tonsil MAITs (Fig. 3B). ImageJ was used to quantify and measure PD1+/- MAIT distance to the edge of

the nearest GC, outlined in Fig. 3C (dashed white line). A total of 349 MAITs from n=5 tonsils were

analyzed with 32% ± 15.9 being PD1+. In comparison, flow results of unstimulated tonsil cells showed

38% ± 14.9 of MAITs to be PD1+, thus confirming the ability of the immunohistochemistry imaging and



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quantification to assess this cell population. PD1+ MAITs were generally located within or closely

surrounding GC’s with a median distance of 66.8 µm from a GC edge compared to PD1- MAITs with a

median distance of 220.2 µm (Fig. 3D). 28.4% of PD1+ MAITs were located within GC’s and thus were

recorded as 0 µm. PD1High expressing MAITs tended to localize with other PD1High cells within GC light

zones. In contrast, PD1low or PD1- MAITs were often found outside of the IgDhigh follicular mantle zone

(Fig 3C-yellow dashed line) and frequently in contact with IgDlow cells. Overall, combined with

phenotypic analysis above, these data reveal PD1+ (and likely CXCR5+) MAIT cells to be Tfh-like in

their proximity to germinal centers.



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Fig. 3. PD1high MAITs preferentially locate near B cell follicles. (A-B) Association between PD1 and CXCR5 expression among tonsil MAITs by flow cytometry. Data were analyzed by Spearman correlation test (A), and two-tailed Mann-Whitney U test (B). *p < 0.05, **p<0.01, ***p<0.001, ****p<0.0001. (C) Location of MAIT cells within tonsils. Representative immunofluorescence images from tonsil cryosections stained with IgD (blue), PD1 (white), CD161 (green), and TRAV1-2 (red) and imaged on a Leica SP8 confocal microscope with 20X objective. Enlarged composite image on right with MAIT cells (TRAV1-2+ CD161+) denoted with arrows. White arrows indicate PD1+ MAITs. Yellow arrows indicate PD1- MAITs. White dashed line outlines germinal center (GC) highlighted by PD1high cells. Yellow dashed line outlines follicular mantle (FM) highlighted by IgD+ naïve B cells. All scale bars represent 100 µm. (D) Quantification of MAIT distance to edge of nearest GC. Tiled images from tonsil donors were visually assessed for co-staining of CD161, TRAV1-2 and then PD1. Distance to nearest GC was then measured using ImageJ. n=359 MAITs were quantified (PD1+ = 103; PD1- = 246) from n=5 tonsil donors. Data are represented as medians with dots representing single MAIT cells. *p < 0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-tailed Mann-Whitney U test.



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Varied transcription factor profile despite similar TCR sequence variability in tonsil and PBMC MAITs

Human MAITs are defined by their invariant TCR-Va chain, with the vast majority (>99%) being Va7.2

or TRAV1-2, though recent studies suggest variability in TRAJ sequence and TRBV usage affects

microbial ligand discrimination, activation, and phenotype (36, 52, 53). Despite this, few studies have

analyzed MAIT TCR sequences from tissue (54–56), with limited sequence analysis in lymphoid organs

(29, 56). Therefore, to determine whether differences in TCR sequence between peripheral and lymphoid

associated MAITs may direct phenotype, we utilized paired TCR-phenotype single-cell Illumina

sequencing as previously described (57). We sorted and single-cell sequenced MAIT cells (identified as

live CD3+ Va7.2+ MR1-Tetramer+ cells) from tonsil samples and PBMCs. We first analyzed TRAJ and

TRBV usage frequency by donor, finding similar inter-donor profiles among tonsil and PBMC MAITs

(Fig. 4A and fig. S3A). In concurrence with previous studies (29, 54, 56, 58), the majority of PBMC and

tonsil MAITs express TRAJ-33 along with TRBV 20-1, 6-1, or 6-4. Although not statistically significant

(when correcting for multiple comparisons), we found an increase in TRAJ 12 and 34 frequency (fig.

S3A), a decrease in TRBV 20-1, and an increase TRBV 7-2 in tonsil versus PBMC MAITs (Fig. 4A).

Furthermore, consensus TRA and TRB sequences, represented using Seq2logo, show little variation

between tonsil and PBMC MAIT CDR3 sequences (fig. S3B). Thus, despite differences in age and

anatomical location, blood and tonsil MAITs share similar TCR usage. Despite significant overlap in

TRAJ and TRBV usage between blood and tonsil MAITs, analysis of TCR sequences suggested higher

number of unique MAIT clones within the tonsil samples compared to PBMCs (Fig. 4B), though this

difference could potentially be attributed to age difference in donors, as older adults tend to have more

clonally expanded MAIT populations (56). In addition to TCR sequence analysis, we further measured, at

the single-cell level, expression of key transcription factors associated with T helper subsets and CD8 T

cells, RORC, TBX21, BCL6, GATA3 and RUNX3, using targeted primers (Fig. 4C). While we found no

difference in the frequency of expression of the Th17 and Th1-associated transcription factors, RORC and



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TBX21, we found an increase in the Tfh lineage defining transcription factor, BCL6, in tonsil relative to

PBMC MAITs. Interestingly, substantial increases in the Th2-assoicated transcription factor, GATA3, and

resident CD8 T cell associated transcription factor, RUNX3 were also observed. These data corroborate

RT-qPCR data showing higher BCL6 and RUNX3 expression in tonsil compared to PBMC MAITs and

MAITfh cells, and similar RORC and TBX21 expression (Fig. 2a, and fig. S1B). No associations were

observed between TRBV usage and transcription factor expression and phenotype (data not shown). These

data suggest that TCR variability is not directly related to MAIT phenotype.

Fig. 4. Overlapping TRBV usage between blood and tonsil MAITs despite transcription factor variance. (A) Heat map of TRBV usage percent frequencies based on Illumina single cell TCR-Phenotype sequencing in tonsil and PBMC MAITs. Each column represents an individual donor (n=4). No statistical significance was found between tonsil and PBMC groups using Multiple t tests comparison accounting for False Discoveries using the Benjamini, Krieger and Yekutieli method. (B) Frequency of unique TCR usage (non-clonally expanded) within PBMC and tonsil MAITs. The frequencies of non-repeating TCR usage based on TRA and TRB sequence per donor were calculated using R. Data are medians. n=4. (C) Targeted single-cell transcription factor expression sequencing in PBMC and tonsil MAITs. Data are represented as frequency of MAIT cells expressing each gene per donor. Data are mean with SD. n³3. *p < 0.033, *p < 0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-tailed Mann-Whitney U test (B) or Multiple students t-tests (C).



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MAITs repopulate mucosal and lymphoid organs following adoptive transfer in TCRa-/- mice

To study the sufficiency of MAITs to provide B cell help in the context of mucosal immune responses in

vivo, we utilized an adoptive MAIT transfer model into Tcratm1Mom/J (TCRa-/-) mice followed by a prime-

boost model of mucosal bacterial infection. Expansion of lung MAITs from WT C57Bl/6 (B6) mice using

an intranasal Salmonella Typhimurium challenge have shown to result in MAIT cells that retain function

even after adoptive transfer (40). TCRa-/- mice have been successfully used in T cell transfer experiments

to study the B cell help potential of non-Tfh CD4 T cells without the potential masking effects of classic

Tfh cells (59). We thus adoptively transferred expanded lung MAITs from B6 into TCRa-/- mice and then

utilized an intranasal prime-boost challenge model with live Vibrio cholerae O1 Inaba (V.c) that induces

systemic and mucosal LPS IgA and IgG responses (60) (Fig. 5A). To confirm MAITs were successfully

transferred into recipient mice, we measured MAIT frequencies and total cell numbers by flow cytometry

of digested lung tissue and spleen (Fig. 5B and fig. S4A-C). The frequency and total cell count of lung

MAITs at Day 42 in MAIT-V.c and MAIT-PBS groups were statistically similar to the WT- V.c group

(Fig 5C and fig. S4B). Importantly, no MAITs were recorded in either Sham-V.c or Sham-PBS group.

Similar MAIT cell counts were also observed in the spleen (fig. S4C), thus demonstrating WT

physiologic MAIT frequencies and repopulation of various organs following adoptive transfer of lung

MAITs. Furthermore, we found no differences between all TCRa-/- groups in total non-MAIT TCRb+ T

cells, thus confirming no significant expansion of contaminating non-MAIT T cells following adoptive

transfer (fig. S4D). We next analyzed the expression of CXCR5 in CD8 T cells, CD4 T cells and MAIT

cells in the WT-V.c and MAIT-V.c groups (Fig. 5D and E). MAITs from both WT-V.c and MAIT-V.c

mice showed no statistical difference in CXCR5 expression when compared to CD4 T cell populations in

lungs suggesting a potential mouse MAITfh population (Fig. 5D and E). Taken together, we found that

adoptive transfer of expanded MAIT cells into TCRa-/- mice repopulates MAIT cells, including CXCR5+

MAITs, in lymphoid and mucosal organs, to levels similar to that of WT mice.



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Fig. 5. Adoptively transferred MAITs expand in lungs of TCRa-/- mice driving B cell differentiation. (A) MAIT adoptive transfer and V.c intranasal challenge time-line. (B) Representative FACS plots of MAITs (TCRb+ MR1-Tetramer+) from lung tissue of WT-V.c (left), MAIT-V.c (middle), and Sham-V.c (right) mice at Day 42 post initial V. cholerae challenge. (C) MAIT frequency as percentage of CD3+. Representative flow cytometry histograms (D) and Mean fluorescence intensity quantification (E) of CXCR5 in non-MAIT CD8 T cells (blue), non-MAIT CD4 T cells (red) and MAIT cells (green) of WT-V.c (left) and MAIT-V.c (right) groups. (F) Frequency of B220+ B cells among live lymphocytes. Frequency of IgD+ naïve B cells (G), IgD- CD27+ CD38+ CD138- Memory B cells (H) and IgD- CD27+ CD38+ CD138++ PB/PCs (I) as percent of B220low-high lymphocytes. Data are represented as Mean with SEM from 4 independent experiments. n= 4-15 mice per group. *p < 0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-tailed Mann-Whitney U test. Legend in black box denotes experimental groups: MAIT-V.c = TCRa-/- plus MAIT Transfer-V.c challenge, MAIT-PBS = TCRa-/- plus MAIT Transfer-PBS challenge, Sham-V.c = TCRa-/- plus Sham Transfer-V.c challenge, Sham-PBS = TCRa-/- plus Sham Transfer-PBS challenge, WT-V.c = WT C57Bl/6J plus Sham Transfer-V.c challenge.

MAIT transfer drives increase in plasmablast differentiation and memory B cell development

We next sought to determine the impact of MAIT adoptive transfer on mucosal B cell differentiation. Day

42 digested lung suspensions were analyzed by flow cytometry with B cells being defined as B220+

lymphocytes. Interestingly, total B220+ cell frequency (Fig. 5F) and total B cell count per lung (fig. S4E)

were decreased in both the MAIT-V.c and MAIT-PBS groups compared to Sham-V.c, Sham-PBS and

WT-V.c groups. Similar decreases in IgD+ naïve B cell frequency were observed in MAIT transfer groups

(Fig. 5G), although no statistical differences were observed in total naïve B cells (fig. S4F). In contrast,

we found that both MAIT-V.c and MAIT-PBS groups had higher Memory B cell (B220+ IgD- CD27mid

CD38+ CD138-) and Plasmablast(PB)/Plasma cell (PC) (B220low IgD- CD27+ CD38+ CD138++)

frequency as a percentage of total B cells compared to Sham-V.c, Sham-PBS and WT-V.c groups (Fig. 5H

and I). Total Memory B cell count per lung was not statistically different between groups with significant

variability observed in the MAIT-V.c group (fig. S4G), though we saw a non-statistically significant

higher total PB/PC cell count per lung in MAIT-V.c compared to Sham-V.c (p=0.097) and WT-V.c

(p=0.179) groups (fig. S4H). Overall, these data suggest that in the absence of ab T cells, transfer of

activated MAITs help induce B cell differentiation from naïve B cells to PB/PCs or memory B cells.



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Increase in V. cholerae specific IgA responses and vibriocidal titer following MAIT transfer

V.c-specific antibodies in blood is a surrogate for protection against cholera (61, 62). Specifically, the

vibriocidal assay, a measurement of complement-fixing bactericidal antibody activity, has been in use

since the mid twentieth century (63) and shown to be the best marker of protection (64, 65). In addition to

the vibriocidal titer, cholera toxin (CT) and lipopolysaccharide (LPS) serum ELISAs have been widely

used to estimate cholera incidence (65–68). The O-specific polysaccharide (OSP) component of LPS and

CT are the immunodominant antigens following cholera infection (69). Therefore, in order to measure the

impact of MAIT transfer on pathogen-specific antibody kinetics we measured serum IgG, IgA, and IgM

antibody responses against whole V.c-lysate, OSP, and CT. We found comparable V.c-lysate IgA

responses in the MAIT-V.c and WT-V.c groups particularly following V.c re-challenge (D28), suggesting

development of a memory response (Fig. 6A). Day 42 endpoint ELISAs reveal a substantial increase in

V.c-lysate IgA responses in the MAIT-V.c group compared to Sham-V.c (p<.0001), and a non-statistically

significant increase (p = 0.58) compared to the WT-V.c group (Fig. 6C). Importantly, minimal V.c-lysate

IgA responses were observed in the Sham-PBS group. MAIT-V.c and MAIT-PBS groups had no

differences in V.c-lysate IgG (Fig. 6B and D) and IgM (fig. S5A and B) responses, despite an increase

compared to Sham transfer groups, thus indicating a non-specific response induced by MAIT transfer.

We saw a non-significant (p=0.054) increase in OSP IgA responses in the MAIT-V.c. group compared to

the Sham-V.c group (Fig. 6E and G), but no differences on OSP-IgG responses (Fig. 6F and H). Notably,

even Sham-infected TCRa-/- mice were able to mount (albeit lower than WT) OSP IgG responses,

indicating that a lack of T cells blunted, but did not eliminate, the ability to mount an IgG response to a

polysaccharide antigen, which is classically T-independent. In contrast, OSP-IgM levels were similar

between the MAIT-V.c, Sham-V.c, and WT-V.c groups, and all were increased compared to MAIT-PBS

and Sham-PBS groups indicating a specific but truly T-independent response (fig. S5E and F). Overall,

very low levels of CT IgA and IgG were observed in all TCRa-/- groups (Fig. 6I-L) compared to the WT-



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V.c group, supporting the role of classical helper T cells in protein antigen B cell help, a response that

adoptive transfer of MAIT cells did not rescue. Analysis of total serum IgA revealed a highly variable but

substantial increase in both MAIT transfer groups over both Sham transfer and WT-V.c groups (fig. S5H).

No statistically significant changes were observed in total IgM and IgG between MAIT-V.c and Sham-V.c

groups (fig. S5G and I).

We further utilized the vibriocidal assay to measure complement-mediated bactericidal activity, the best

studied and most commonly used correlate of protection from cholera in humans (64, 65). We found an

increase in vibriocidal titer in the MAIT-V.c group compared to the Sham-V.c group (p=0.069), and no

difference when compared to the WT-V.c group, suggesting MAIT transfer is sufficient to rescue

vibriocidal responses (Fig. 6M). Unlike the ELISAs, no background vibriocidal responses were observed

in the MAIT-PBS group demonstrating the specificity of this assay. Taken together, adoptive transfer of

MAIT cells into T-cell deficient mice rescued deficiencies in V. cholerae-specific IgA and vibriocidal

antibody responses.



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Fig. 6. MAITs promote V. cholerae-specific IgA and protective vibriocidal antibody responses in TCRa-/- mice. Serum ELISAs against V. cholerae whole lysate, OSP and CT. (A-D) IgA and IgG V. cholerae lysate ELISAs from weekly cheek bleed (A & B) and Day 42 endpoint serum (C & D). (E-H) IgA and IgG OSP ELISAs from weekly cheek bleed (E & G) and Day 42 endpoint serum (F & H). (I-L) IgA and IgG CT ELISAs from weekly cheek bleed (I & J) and Day 42 endpoint serum (K & L). Data are represented as ELISA units normalized to a pooled serum positive control from WT B6 mice challenged with V. cholerae. (M) Vibriocidal titer of Day 42 endpoint serum. Data are represented as Mean with SEM from 5 independent experiments. n= 6-22 mice per group. *p < 0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-tailed Mann-Whitney U test. Legend in black box denotes experimental groups.



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Discussion

MAIT cells are widely appreciated for their ability to respond rapidly to microbial antigens and cytokines

through potent production of Th1 and Th17-associated inflammatory cytokines and cytotoxic molecules.

Recent studies have highlighted the capacity of human MAITs to provide B cell help in vitro, and

promote B cell differentiation and antibody production in animal models (23, 25, 26). Despite these

findings, whether there remains a pre-defined MAIT subset capable of B cell help remains uncertain.

Additionally, many studies have detected MAITs within mucosal lymphoid tissues but have yet to

understand their role in these environments (33, 41, 70). In this study we begin to define the role of

MAITs in human mucosal lymphoid organs and uncover a novel T follicular helper-like MAIT population

defined by CXCR5. This MAITfh subset highly expresses PD1, ICOS, BCL6, and IL-21, and

preferentially locates near germinal centers. We further add to the body of literature proving the ability of

MAITs to provide B cell help in vivo through sufficiency experiments following mucosal challenge. Here

we show that adoptively transferred MAITs can rescue protective microbe specific responses, and

promote B cell differentiation in the absence of other ab T lymphocytes.

We report a subset of CXCR5+ MAIT (MAITfh) cells that are enriched in mucosal lymphoid organs and

represent a small percentage of peripheral blood MAITs (Fig. 1C). We further report that MAITfh cells

highly express the B cell help co-stimulatory molecules, PD1 and ICOS, compared to CXCR5- MAIT

cells (Fig 1D and E). In addition, we show high relative expression (»20-70 fold) of CD40L in both

MAITs and MAITfh groups compared to the control CD8+ population (fig. S1B). PD1, ICOS and CD40L

expression are a hallmark of GC Tfh cells (71). PD1, although long considered a marker of T cell

exhaustion, has an important role in Tfh GC positioning and function (5). ICOS and CD40L engagement

with ICOS Ligand and CD40 expressed on B cells is essential for GC formation and Tfh activation (6, 7).

CD40L is also implicated in MAIT induced maturation of dendritic cells (72). Based on the Tfh

costimulatory molecule profile, we hypothesized that MAITfh cells may share further transcriptional



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similarities with Tfh cells. In support, we found that MAITfh cells in tonsils and PBMCs have higher

BCL6 expression than MAITs and Tfh cells in both PBMCs and tonsils (Fig. 2A). Additionally, we

reported more BCL6+ MAITs in tonsils relative to PBMC MAITs (Fig. 4C). BCL6 is considered the

master regulator of the Tfh lineage (73) and is similarly required for iNKTfh B cell help responses (18).

Furthermore, expression of BLIMP1, which is a negative regulator of Tfh differentiation (73) was lower

in the tonsil MAITfh vs MAIT groups. Although further research into the transcriptional regulation of

MAITfh cells is necessary to understand the requirements for differentiation, plasticity and function, these

data support a B cell help transcriptional phenotype among the MAITfh subset.

We report MAITfh expression of the B cell help cytokines IL-21, IL-10, IL-6 and BAFF (Fig. 2 and fig.

S2). IL-21 and IL-10 were specifically upregulated in MAITfh cells, while BAFF and IL-6 were also

highly expressed in both PBMC and tonsil MAIT cells compared to CD8 controls. IL-21 plays an

important role in T dependent B cell differentiation, class switching and antibody production (74) and

together with IL-6 promotes Tfh differentiation (75). IL-10 promotes antibody production and class

switching (76). IL-21 expression in MAITs has been demonstrated in low concentrations in PBMC

MAITs (23), and in a subset of PD1High CXCR5- MAITs in pleural effusions from Tuberculosis patients

(24). This study also presents a small subset of CXCR5+ PD1high MAITs, though they do not investigate

whether this population expressed IL-21 as well (24). In contrast, we show that tonsil CXCR5+ MAITs

are largely IL-21+ IFNg- (Fig. 2G) and produce significantly more IL-21 than CXCR5- MAITs (Fig. 2H).

It is unclear whether PD1High CXCR5- MAITs in pleural effusions are transcriptionally or developmentally

related as potential precursors to CXCR5+ MAITs found in lymphoid tissues. Furthermore, the B cell

survival cytokine, BAFF, is integral in T independent class switching and differentiation (77), and is

similarly produced by iNKTfh cells to promote B cell responses (18). Thus, it is possible MAITfh cells

can aid in B cell help through cognate interactions via direct MR1-TCR, co-stimulatory molecule

engagement and cytokine expression, or through non-cognate interactions such as promoting Tfh

differentiation or licensing of dendritic cells.



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We have observed that PD1high MAITs preferentially locate within or around GCs in human tonsils (Fig.

3C and D). We further prove these cells are likely CXCR5+ based on high co-expression with PD1 (Fig.

3A and B). This is noteworthy since spatial positioning of T and B cells within lymphoid tissues is critical

in understanding their phenotype. For instance, CXCR5+ PD1High GC Tfh cells reside primarily in the light

zone of GCs where they promote affinity maturation and differentiation of GC B cells leading to high

affinity antibody responses (71). Thus, by combining imaging results with in vitro MAITfh expression of

B cell help co-stimulatory markers and cytokines (Fig. 1 and 2), we can conclude that MAITfh cells share

both phenotype and positioning with traditional GC Tfh cells. Thus we hypothesize that this population

may be involved in cognate or contact-dependent B cell help as is seen in other innate Tfh subsets, such

as iNKTfh (12). Though as this population is relatively small in tonsils, further research will need to

address the in vivo relevance to overall mucosal antibody production, the role of MAITfh-B cell

interactions in affinity maturation, and development of B cell memory.

We report that adoptive transfer of murine MAITs boosts pathogen-specific antibody responses in the

absence of other ab T lymphocytes. We further previous work on MAIT-B cell help responses in animal

models (25, 26), by using an adoptive transfer model to show that MAIT cells are sufficient to increase

functional anti-bacterial antibodies in the context of a mucosal challenge. Most notably, we show that

adoptive transfer of MAIT cells rescues the deficiency in V.c-specific IgA and vibriocidal antibodies of

infected TCRa-/- mice when compared to infected WT mice. Examination of antibody kinetics of V.c

specific IgA in mice receiving MAIT transfer suggest a memory phenotype, based on substantial

increases on Day 35 (one-week post re-challenge) (Fig. 6A), and is associated with higher lung PB/PC

and memory B cell frequencies (Fig. 5F and G). Interestingly, we also report MAIT-V.c mice had

significant systemic V.c specific IgA and total IgA responses compared to Sham-V.c mice (Fig. 6A and

fig. S5A). These data may be a result of the mucosal nature of the intranasal challenge, which has shown



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to induce both mucosal and systemic immunity in other bacterial infections (78). Notably, increases in

total IgA in MAIT-PBS mice suggests MAITs may specifically promote IgA class switching. In support,

pulmonary MAITs in mice have recently been shown to produce both IL-10 (79) and TGF1-b (80), key

cytokines in IgA class switching (81). Although CXCR5 staining of lung MAITs suggests a similar Tfh-

like MAIT phenotype in mice (Fig. 5C and D), limitations of available reagents for identifying murine

MAITs by IHC precludes our ability to confirm a MAITfh subset in mice. Future analysis of lung and

lymph node MAITs is necessary to confirm a MAITfh subset in mice and its potential role in IgA class

switching and antibody production. Given that systemic V. cholerae specific IgA and vibriocidal

responses strongly correlate with protection against subsequent cholera re-infection (62, 64, 65), these are

promising data in the context of mucosal vaccines, where IgA is often the primary means of protection,

and development of long-lasting protective memory responses is limiting (82).

In addition to promoting a protective antibody response, we report amplification of non-specific

antibodies following MAIT transfer. We report similar V. cholerae lysate IgG (Fig. 6D) and IgM (fig.

S5B), total IgA responses (fig. S5H), and similar changes in B cell subset frequencies in our MAIT-PBS

and MAIT-V.c groups (Fig. 5E-H). Similar MAIT amplification of non-specific responses were reported

in non-human primates (26) and FcgRIIB-/- mice (25). Specifically, Rahman et al. reported increased total

IgM and IgG antibody production and increased IgD- B cells (indicating class switching) when non-

human primate MAITs were co-cultured with B cells. Murayama et. al 2019 show increases in total IgG

and anti-dsDNA IgG and IgA when MAITs were co-cultured with B cells from FcgRIIB-/- mice. They

also report reduction in anti-dsDNA IgG in MR1-/- (MAIT deficient) FcgRIIB-/- mice, though total IgG

was not published. It should be noted that in all studies, MAITs were either activated in vitro in direct co-

culture, or in vivo in the absence of regulatory T cells (TCRa-/- mice). Therefore, such responses may be a

result of lack of either MAIT and/or B cell regulation leading to aberrant antibody responses. This has

interesting consequences in the setting of mucosal immunity and in autoimmunity. In a setting of proper T



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and B cell regulation, MAITs may help to promote anti-microbial responses and therefore could

potentially be targeted as a vaccine adjuvant. Conversely, in a dysregulated environment, such as in

FcgRIIB-/- or TCRa-/- mice, MAITs may enhance aberrant antibody production against self or commensal

microbes thus enhancing the pro-inflammatory autoimmune state. These aberrant responses open many

questions into cell extrinsic MAIT regulation which remains relatively unstudied.

There are a number of limitations that need to be addressed when considering the results of this study. In

our human in vitro experiments, we compare PBMCs from healthy adults ages 18+ to tonsils from

pediatric patients, ages 2-16, undergoing tonsillectomy. MAITs expand immediately after birth and reach

adult frequencies by approximately 10 years of age, and most reach maturity (CD45RO+) by 6 months of

age (83). Therefore, it is possible that age may explain low MAIT percentage in tonsil samples, though

we do not anticipate age-associated phenotypic effects. Secondly, the low sample size and lack of paired

blood and tonsil samples significantly limits our TCR analyses (Fig. 4), and thus our observations

regarding lack of differences in TCR usage between tonsil and blood MAITs, may be underpowered.

Regarding our animal studies, a limitation of this study is the lack of mucosal antibody measurement as

we recorded only systemic responses in serum. While mucosal secretory IgA (sIgA) and circulating IgA

responses may differ, measurement of systemic antibodies is standard practice for mucosal vaccine

response testing in humans, and V.c specific serum IgA response and vibriocidal titer strongly correlate

with protection in humans (62, 64, 65). Thus, these serum responses are likely relevant to mucosal

protection.

In conclusion, we identify a Tfh like MAIT population enriched near germinal centers of mucosal

lymphoid tissues that express cytokine and transcriptional profiles consistent with B cell help. In addition,

we show in an animal model that MAITs are sufficient to rescue pathogen-specific functional antibody

responses following mucosal challenge in the absence of other classical T cells. Our work strengthens a

growing body of evidence supporting the capacity of MAITs to aid in B cell help and antibody mediated



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immunity, and support the expanding phenotypic heterogeneity of MAITs. As MAITs are enriched at the

mucosa and can be activated with specific ligands, we hypothesize that they may be a promising target to

enhance mucosal vaccines.



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Materials and Methods

Peripheral blood and tonsil collection

Healthy adult blood was collected from residual leukocyte packs following blood donation (ARUP,

Sandy, UT), and PBMC’s were isolated by density gradient centrifugation (Lymphoprep, StemCell

Technologies). Residual palatine tonsil samples were acquired from pediatric patients, ages 2-16,

undergoing routine tonsillectomies for tonsillar hyperplasia or recurrent tonsillitis. All tonsil samples

were stored on ice in complete media (RPMI 1640 with 10% FBS, 1% penicillin/streptomycin, and 15

mM HEPES) (R10) and processed within 2 hours of surgery. 1 cm x 1 cm pieces were cut and frozen in

O.C.T. Compound (Fisher) at -80°C for later use. The remaining tonsil tissue was minced in media and

dissociated using a 100 µM cell strainer. Tonsil mononuclear cells were isolated by density gradient

centrifugation (Lymphoprep, StemCell Technologies). PBMC and tonsil mononuclear cells were frozen

in media containing 25% FBS and 10% DMSO at -80°C for later use. All human samples were de-

identified prior to receipt, and the research protocol was deemed exempt by the University of Utah

Institutional Review Board (Protocol 100683).

Flow cytometry and cell sorting

Prior to surface staining, human and mouse single cell suspensions were labeled with Fixable Viability

Dye eFluor 780 (eBioscience) according to manufacturer’s protocol to delineate live cells. Cells were

washed and incubated with fluorochrome conjugated antibodies for 20 min at room temperature (RT).

Mouse cells were incubated in anti-mouse CD16/CD132 (Fc block) for 15 minutes prior to surface

staining. Mouse and human MR1-Tetramers (NIH Tetramer Core) diluted 1:400 were added to surface

stain. Prior to mouse MR1-Tetramer staining, cells were incubated for 15 minutes with unlabeled i6FP-

Tetramer (NIH Tetramer Core) to minimize non-specific binding. For intracellular cytokine staining, cells

were fixed and permeabilized using Foxp3/Transcription Factor Staining Buffer set (eBioscience)

according to manufacturer’s protocol and incubated with fluorochrome conjugated antibodies for 40 min



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at RT. Cells were analyzed using BD LSR Fortessa for phenotypic analyses, or Aria II (BD Biosciences)

for cell sorting. The following monoclonal antibodies to human were used : CD3- AF700 (clone OKT3),

CD8-FITC/BV605 (cloneSK1, TCR Va7.2-BV711/PE-Cy7 (clone 3C10), PD1-BV605/AF700 (clone

EH12-2H7), ICOS-PerCP-Cy5.5/BV605 (clone C398.4A), CXCR5-PE-Cy7/APC (clone J252D4), IL-21-

APC (clone 3A3-N2), IFNg-BV421 (clone B27), IL-6-FitC (clone MQ2-13A5), IL-10-PerCP-Cy5.5

(clone JES2-9D7), CD161-AF488 (clone HP-3610) (Biolegend), CD4-BUV496 (clone SK3), CD3-

BUV395 (clone SK7) (BD Biosciences). The following monoclonal antibodies to mouse were used: CD3-

PE-Dazzle594 (clone 17A2), TCRb-BV421 (clone H57-597), CXCR5-BV605 (clone L138D7), CD44-

BV650 (clone IM7), TCRgd-PE-Cy7 (clone GL3), CD8-APC (clone 53-6.7), CD45R-PE-Cy5 (clone

RA3-6B2), IgD-BV510 (clone 11-26c.2a), CD138-BV605 (clone 281-2), CD38-PE-Cy7 (clone 90,

CD27-APC (clone LG-3A10) (Biolegend), CD4-FITC (clone GK1.5) (Tonbo Biosciences).

In vitro stimulation assays

For flow cytometry intracellular cytokine staining, 1-2 x 106 mononuclear cells isolated from blood or

tonsil samples were cultured in a round bottom 96 well plate at 37°C 5% CO2 in R10 media for 6 hours

with or without 200 ng/ml phorbol myristate acetate (PMA) and 1.0 µg/ml Ionomycin (Sigma-Aldrich).

Brefeldin A Solution (Biolegend) was added at 1X for the final 4 hours. For measurement of IL-21

production, tonsil mononuclear cells were sorted into CD8+ CXCR5-, CD4+ CXCR5+ PD1High, MR1-Tet+,

Va7.2+, CXCR5-, MR1-Tet+, Va7.2+, CXCR5+ populations and 4x104 cells in R10 media were cultured

in a round bottom 96 well plate for 48 h with 20 ng/ml PMA and 1.0 µg/ml Ionomycin as described in

Shen et al (48). Plates were centrifuged and cell supernatant was isolated for ELISAs.

qRT-PCR

PBMC and tonsil CD8+ CXCR5-, CD4+ CXCR5+, MR1-Tet+ Va7.2+ CXCR5-, MR1-Tet+ Va7.2+

CXCR5+ cells were sorted into TRIzol Reagent (Qiagen). Total RNA was isolated using the RNeasy



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Micro kit and cDNA synthesized a High-Capacity cDNA Reverse Transcription Kit (Applied

Biosystems). Targeted qPCR was performed using the QuantStudio 6 Real-Time PCR system using the

following Taqman Primers (Applied Biosystems): BCL6 (Hs00153368_m1), Tnfsf13b

(Hs00198106_m1), IL6 (Hs00174131_m1), IL10 (Hs00961622_m1), IL21 (Hs00222327_m1), TNFa

(Hs00174128_m1), TGFB1 (HS00998133_m1), Tcf7 (Hs01556515_m1), Tbx21 (Hs00894392_m1),

Runx3 (Hs01091094_m1), Rorc (Hs01076112_m1), Perforin (Hs00169473_m1), Pdcd1

(Hs01550088_m1), Il17a (Hs00174383_m1), Ifng (Hs00989291_m1), Icos (Hs00359999_m1), Gzmb

(Hs00188051_m1, Cxcr5 (Hs00173527_m1), Cd40l (Hs00163934_m1), Cd38 (Hs01120071_m1),

Blimp1 (Hs00153357_m1), Ascl2 (Hs00270888_s1), ActB (Hs99999903_m1).

ELISAs and Vibriocidal assay

IL-21 concentration from tonsil mono-cultures was measured using Human IL-21 ELISA MAX Deluxe

kit (Biolegend) according to manufacturer’s protocol. Serum V.c Lysate, OSP:BSA and CT ELISAs were

performed as follows. Nunc Maxisorp flat bottom 96 well plates (Invitrogen) were coated with V.c Lysate

or OSP:BSA (gift from Dr. Edward Ryan (Massachusetts General Hospital, Boston, MA) (1.0 µg/ml ) in

PBS and incubated overnight at 4°C. For CT ELISAs, Nunc plates were first coated with

Monosialoganglioside GM1 (Sigma-Aldrich) (1.0 µg/ml) in 50mM carbonate buffer overnight at 4°C.

Plates were subsequently blocked with 1% bovine serum albumin (BSA) (Sigma-Aldrich) and incubated

overnight with CT (Sigma-Aldrich) (2.5 µg/ml) in carbonate buffer. All plates were then blocked with 1%

BSA and 25 µl of mouse serum diluted 1:10 in 0.1% BSA-0.5% Tween 20 in PBS (BSA-PBST) was

added for 2 hours. Plates were incubated with anti-mouse IgG (Invitrogen), IgA (Invitrogen), or IgM (Life

Technologies) HRP conjugate diluted 1:1000 in BSA-PBST for 2 hours. 100 µl of TMB substrate

(Thermo Fisher) was added and plates were immediately read kinetically at 405 nm (7 min x 1 min

intervals) using plate reader (Biotek). ELISA measurements were recorded as max interval slope and

normalized to pooled positive mouse sera included on each plate. Total Ig ELISAs were performed as



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described above except Nunc plates were pre-coated with anti-mouse IgA (clone RMA1), anti-mouse IgG

(clone Poly 4053), or anti-mouse IgM (clone RMM-1) (Biolegend) and standard curves were generated

using 2-fold dilutions of purified Mouse IgA, IgM and IgG (Southern Biotech). Plates were incubated

with corresponding anti-mouse HRP antibodies and TMB substrate was neutralized after 10 min with 0.2

N HCl. Plates were read at 605 nm and concentrations were calculated based on the standard curve.

Vibriocidal titer was measured as described (84). Vibrio cholerae serotype 01 Strain El Tor Inaba N16961

grown in LB media was used as target organism for assay, and serum samples were diluted to a starting

concentration of 1:5.

Immunofluorescence staining

Unfixed pediatric tonsil samples frozen in OCT medium were cut into 9µm thick sections using a cryostat

(Leica) and stored at -80°C. Tonsil sections were brought to room temperature and then fixed at -20°C for

10 min in pre-cooled 50:50 Acetone:Methanol solution. Sections were dried and then rehydrated in PBS

for 5 min with gentle shaking. The sections were then blocked in 2% normal mouse serum (Invitrogen)

for 1 hour and subsequently stained overnight at 4°C with mouse anti-human TCR Va7.2-PE (Clone

3C10, Biolegend), mouse anti-human CD161-AF488 (Clone NKR-P1A, Biolegend), mouse anti-human

IgD-BV421 (Clone IA6-2, Biolegend) and mouse anti-human PD1-APC (Clone EH1202H7, Biolegend)

all diluted 1:100. Sections were washed 3x in PBS and mounted using ProLong Gold Antifade Mounting

media (Thermo Fisher). Full tonsil cross section images were acquired using the Leica Sp8 Confocal

microscope and then processed and analyzed using ImageJ.

Single cell TCR sequencing

MAITs from PBMC’s and tonsils were single cell sorted using the Aria II cell sorter (BD Biosciences)

directly into One Step RT-PCR reaction mix (NEB) loaded in MicroAmp Optical 96-well reaction plates

(Applied Biosystems). MAITs were defined as CD3+ Va7.2+ MR1-Tetramer+ cells. Following reverse



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transcription and preamplification reaction, a series of three nested PCR’s were run using primers for

TCR sequence and gene expression as described (57). Subsequent sequencing data analysis was

performed as described (57).

Bacterial culture and V. cholerae lysate preparation

The attenuated Salmonella Typhimurium strain BMM50 was a gift from Dr. Stephen McSorely (UC

Davis, Davis, CA). BMM50, like previously published S. Typhimurium BRD509 (39), has a deleted aroA

gene and intact riboflavin pathway. Vibrio cholerae serotype 01 Strain El Tor Inaba N16961 was a gift

from Dr. Edward Ryan (Massachusetts General Hospital, Boston, MA). All strains were inoculated from

single colonies into Luria Bertani (LB) broth shaking at 220 RPM 37°C overnight. They were then

subcultured 1:10 in fresh LB and incubated for 3-4 hours shaking at 37°C until log phase was reached.

Cultures were washed 2x in sterile PBS and normalized to 0.1 OD600 using a microplate absorbance

spectrophotometer (BioRad). V. cholerae at 0.1 OD600 was used as inoculum for intranasal challenge

model. S. Typhimurium BMM50 was diluted 1:10 with PBS to an OD600 of 0.01 for adoptive transfer

inoculum. V. cholerae lysate for ELISAs was prepared as follows. V. cholerae was cultured from a single

colony in LB broth shaking at 220 RPM 37°C overnight. V. c culture was then pelleted and washed 3x

with cold sterile PBS. Following resuspension in PBS, sample was sonicated at 40% power 4 x 1 min

intervals, keeping on ice for 1 minute between runs. Sonicated sample was then centrifuged at 15000 rpm

x 10 min at 4°C, and the supernatant removed and sterilized using 0.2 µm syringe filter (Thermo Fisher).

Protein content was measured using 260/280 absorbance ratio with Take3 Micro-Volume Plate reader

(BioTek)

Mice

Tcratm1Mom/J mice (85) (on a B6 background) were obtained from The Jackson Laboratory (Bar Harbor,

ME) and bred at the University of Utah Comparative Medicine Center mouse facility. WT C57BL/6J



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mice were obtained directly from The Jackson Laboratory prior to the start of the experiment. 6-10-week-

old female and male mice were used in adoptive transfer studies. All mice were housed under specific

pathogen-free conditions. Experiments were performed in strict accordance with the NIH guide for the

Care and Use of Laboratory animals and institutional guidelines for animal care at the University of Utah

under approved protocol #17-01011.

Adoptive transfer

Adoptive transfer of pulmonary MAITs was adapted from protocol described (40). C57BL/6 mice were

infected i.n. with 106 CFU S. Typhimurium BMM50 suspended in 50 µl sterile PBS (25 µl per nares).

Mice were rested for 7 days to allow for MAIT expansion, in which mice were weighed daily and

evaluated for signs of clinical pneumonia symptoms (respiratory distress, inactivity, ruffled fur, hunched

posture). Mice with loss of >20% of body weight and/or severe clinical symptoms were euthanized. On

day 7, mice were euthanized and the lungs were perfused with 10 mls of PBS through the heart and then

removed. Single cells suspensions of lungs were prepared using the gentleMacs Lung Dissociation kit,

mouse (Miltenyi Biotech) according to the manufacturer’s protocol. Red blood cells were lysed using

ACK Lysis buffer (Thermo Fisher), and single cell suspensions were stained and sorted using the BD

FACS Aria II. MAITs were defined as live CD3+ B220- TCRgd- CD44high TCRb+ MR1-Tetramer+

lymphocytes. Approximately 5x104 - 1x105 MAITs were sorted per mouse. 105 MAITs suspended in 100

µl was transferred via retroorbital injections into lightly anesthetized mice. Transferred mice were

monitored to confirm recovery following injections, and rested for 2 weeks to allow for MAIT expansion

before use in intranasal challenge model.

Intranasal challenge model

The following protocol was adapted from Nygren et al (60) to induce V. cholerae specific antibody

responses through a prime-boost live bacterial model. In brief, on Day 0 mice were anesthetized using



https://doi.org/10.1101/2020.10.05.326488


isoflurane and infected i.n. with 106-107 CFU V. cholerae 01 Biotype El Tor Serotype Inaba suspended in

50 µl sterile PBS (25 µl per nares). Mice were monitored for 7 days post infection for weight loss and

clinical signs of pneumoniae (outlined above). Mice with loss of >20% of body weight and/or severe

clinical symptoms were euthanized. Mice were subsequently re-challenged on Day 28 using the above

protocol. Blood samples were collected weekly, prior to infections on Day 0 and 28, from submandibular

bleeds on lightly isoflurane anesthetized mice. Blood serum was isolated using BD Microtainer SST tubes

(BD Biosciences) according to the manufacturer’s protocol. On Day 42 (2 weeks post re-challenge), mice

were euthanized using a bell jar and isoflurane. Blood was collected immediately via cardiac puncture and

serum isolated. Lungs were perfused and single cell suspensions were processed as described above.

Spleens were excised, ground through 70 µm filters in cold R10 media. Red blood cells were lysed using

ACK Lysis Buffer (Thermo Fisher). 1-2x106 cells from lung and spleen cell suspensions were stained

with fluorochrome conjugated antibodies for flow cytometric analyses.

Statistical analysis

All statistical tests were performed using Prism version 8.4.2 (GraphPad Software, La Jolla, CA, USA).

Differences were compared using two-tailed Mann Whitney U tests, or Multiple t tests comparisons

accounting for False Discoveries using the Benjamini, Krieger and Yekutieli correction as indicated.

Graphs were created using Prism or R (Vienna, Austria) and the graphing package ggplot2 (H. Wickham,

Springer-Verlag, NY, USA).



https://doi.org/10.1101/2020.10.05.326488


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Acknowledgements

Funding: This research was supported by the National Institutes of Health (R01 AI130378 to D.T.L. and

T32 AI138945 to O.J.). This work was also supported by the University of Utah Flow Cytometry Facility

in addition to the National Cancer Institute through Award Number P30CA042014. We would also like to

thank the staff of the Office of Comparative Medicine, and University of Utah Cell Imaging Core.

Author Contributions: O.J. and D.T.L. designed and directed the project. O.J and S.T. performed the

experiments. K.F. and J.S.H. contributed to experimental methodology. J.D.M. provided samples and

resources. D.T.L acquired funding. O.J. and D.T.L wrote the paper, and all authors discussed results,

provided critical feedback, and contributed to the final manuscript.

Declaration of interests: None.



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Supplementary Materials

Supplementary Fig. 1. qPCR and flow gating strategy (Fig 2) and additional FAC sorted qPCR targets. Representative flow cytometry gating strategy for qPCR FAC sorted and flow populations in Fig.2 from (A) PBMC and (B) tonsils. (C) Heat map of additional qPCR targets described in Fig 2A-E. As described, data are represented as DDCt relative to b-Actin and then the PBMC-CD8+ CXCR5-

population. Rows denote gene ID and columns denote sample group (P=PBMC, T=tonsil). Heat map data are represented by mean of n=4 samples per group.



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Supplementary Fig. 2. High IL-21 baseline expression in unstimulated CXCR5+ MAITs in tonsils. (A) Representative flow cytometry from 6 hour no stimulation controls for PBMC and tonsil T cell populations showing IL-21 and IFNg co-expression. (B) Frequency of T cell populations expressing IL-21, IFNg or both. Data are represented median from two independent experiments. n³5. Flow cytometry data showing frequency of T cell subsets expressing IL-10 (C) and IL-6 (D) following in vitro stimulation with 6 h PMA/Ionomycin. Data are represented as mean with SEM from 2 independent experiments. n=8. *p < 0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-tailed Mann-Whitney U test. Legend in black box denotes experimental groups.



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Supplementary Fig. 3. PBMC and tonsil MAIT TRAJ usage frequency and TRA-TRB Seq2logo plots. (A) Heat map of TRAJ usage percent frequencies based on Illumina single cell TCR-Phenotype sequencing in tonsil and PBMC MAITs. Each column represents an individual donor (n=4). No statistical significance was found between tonsil and PBMC groups using Multiple t tests comparison accounting for False Discoveries using the Benjamini, Krieger and Yekutieli method. (B) Visual depiction of consensus CDR3a (left) and CDR3b (right) amino acid sequences from PBMC (top) and tonsil (bottom) MAITs. Analysis was confined to first 12 and 13 amino acids for CDR3a and CDR3b, respectively.



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Supplementary Fig. 4. Flow gating for murine lung MAITs and B and T lymphocyte counts. (A) Representative flow gating of murine MAITs from digested lung tissue highlighted in Fig. 5a. MAITs were defined as live B220- CD3+ TCRgd- CD44High TCRb+ MR1-Tetramer+. Only WT-V.c., MAIT-V.c., and Sham-V.c. groups are portrayed. (B) Total MAIT count per lung. (C) Total MAIT cell count per spleen. (D) Total non-MAIT CD3+ TCRb+ T cells per lung. (E) Total B220+ B cells per lung. (F) Total IgD+ Naïve B cells per lung. (G) Total IgD- CD27+ CD38+ CD138- Memory B cells per lung. (H) Total IgD- CD27+ CD38+ CD138++ PB/PCs per lung. Data are represented as Mean with SEM from 4 independent experiments. n= 4-15 mice per group. *p < 0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-tailed Mann-Whitney U test. by two-tailed Mann-Whitney U test. Legend in black box denotes experimental groups.



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Supplementary Fig. 5. Serum IgM and total IgM, IgG and IgA ELISAs. (A-F) Serum V. cholerae lysate, OSP, and CT IgM ELISAs representing weekly antibody kinetics (A, C & E) and Day 42 endpoint responses (B, D & F). Data are represented as ELISA units normalized to a pooled serum positive control from WT B6 mice challenged with V. cholerae. Total IgM (G), IgA (H) and IgG (I) ELISAs from Day 42 endpoint serum. Total Ig was quantitated using a standard curve based on known Ig quantities. Data are represented as Mean with SEM from 5 independent experiments. n= 6-22 mice per group. *p < 0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-tailed Mann-Whitney U test. by two-tailed Mann-Whitney U test. Legend in black box denotes experimental groups.



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